[go: up one dir, main page]

WO2020262107A1 - Procédé de production de (r)-réticuline - Google Patents

Procédé de production de (r)-réticuline Download PDF

Info

Publication number
WO2020262107A1
WO2020262107A1 PCT/JP2020/023561 JP2020023561W WO2020262107A1 WO 2020262107 A1 WO2020262107 A1 WO 2020262107A1 JP 2020023561 W JP2020023561 W JP 2020023561W WO 2020262107 A1 WO2020262107 A1 WO 2020262107A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
nucleotide sequence
protein
reticuline
enzymatic activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2020/023561
Other languages
English (en)
Japanese (ja)
Inventor
中川　明
博道 南
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ishikawa Prefectural University
Original Assignee
Ishikawa Prefectural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ishikawa Prefectural University filed Critical Ishikawa Prefectural University
Priority to JP2021528257A priority Critical patent/JP7648038B2/ja
Priority to EP20832173.7A priority patent/EP3988663A4/fr
Priority to US17/622,288 priority patent/US12359233B2/en
Publication of WO2020262107A1 publication Critical patent/WO2020262107A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01037—5-Aminolevulinate synthase (2.3.1.37)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to a method for producing (R) -reticuline, which is a plant benzylisoquinoline alkaloid.
  • Opioid analgesics such as morphine are known as pharmaceutically useful compounds belonging to benzylisoquinoline alkaloids.
  • opioid analgesics have been produced by extraction from natural plants. Analgesics obtained by this method utilizing secondary metabolites of plants are more expensive than analgesics produced by other methods, and therefore another method has been desired.
  • Some benzylisoquinoline alkaloids, including morphine, can be totally synthesized by chemical synthesis. However, due to the complex structure and chirality of alkaloids, it is difficult to produce them at low cost.
  • Patent Document 1 Non-Patent Documents 1 and 2.
  • This method which uses microorganisms as host cells, combines plant and microbial enzymes to reconstruct the isoquinoline alkaloid biosynthetic pathway.
  • benzylisoquinoline alkaloids are synthesized from tyrosine in Magnolia family, Ranunculaceae, Berberidaceae, Papaveraceae, and many other plant species, and most of them use (S) -reticuline as a biosynthetic intermediate.
  • Thebaine which is a raw material for opioid analgesics, is obtained through a reaction in which (S) -reticuline is converted to the optical isomer (R) -reticuline.
  • the enzyme that converts (S) -reticuline to (R) -reticuline has not been identified for a long time, but in Non-Patent Document 3, STORR is an enzyme that converts (S) -reticuline to (R) -reticuline.
  • a ((S) -t Economics- (R) -reticuline) protein was identified (Non-Patent Document 3).
  • Non-Patent Document 3 does not disclose a specific method for producing (R) -reticuline from (S) -reticuline using a microorganism.
  • Patent Document 2 discloses a method for producing (R) -reticulin using a eukaryote such as yeast as a host cell and using the same or similar amino acid sequence as STORR (Patent Document 2). ..
  • Patent Document 2 does not disclose a method for producing (R) -reticuline using a prokaryote as a host cell. This is because it is difficult to functionally express the P450 enzyme and the saltazine synthase (SalS) in prokaryotes. Therefore, in Patent Document 2, the target applicable as a host cell is limited to eukaryotes such as yeast, and it is difficult to produce (R) -reticrine using a prokaryote (for example, Escherichia coli) as a host cell.
  • a prokaryote for example, Escherichia coli
  • an object of the present invention is to provide a method for producing (R) -reticuline from (S) -reticuline with high conversion efficiency. It is also an object of the present invention to provide a method capable of producing (R) -reticuline with high conversion efficiency even when a prokaryote is used as a host cell. Another object of the present invention is to supply an inexpensive opioid analgesic by constructing a thebaine production system applicable to Escherichia coli, which is a practically useful prokaryote.
  • the present inventor has found that (R) -reticuline can be obtained with extremely high conversion efficiency by adopting the method of the present invention with respect to the above-mentioned problems. It was also found that the method of the present invention makes it possible to produce (R) -reticuline with extremely high conversion efficiency even when a prokaryote is used as a host cell. In the method of the present invention, the amount of (S) -reticuline was finally below the detection limit, and the ratio of (R) -reticuline produced was apparently 100% of the total amount of reticuline.
  • the method for producing (R) -reticuline of the present invention comprises a DNA having a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 2 and encoding a protein having the enzymatic activity of CYP80Y2. It consists of a certain gene 1 (hereinafter, also referred to as "gene encoding CYP80Y2") and a nucleotide sequence having 70% or more homology with respect to the nucleotide sequence of SEQ ID NO: 3, and exhibits the enzymatic activity of oxidoreductase.
  • a gene 2 (hereinafter, also referred to as “gene encoding oxidoreductase”), which is a DNA encoding a protein having a protein, into a host cell to obtain a recombinant host cell, and in the recombinant host cell.
  • a step of expressing the protein having the enzymatic activity of CYP80Y2 and the protein having the enzymatic activity of the oxidoreductase, and a step of producing (R) -reticulin from (S) -reticulin by the recombinant host cell It is characterized by including.
  • the method for producing (R) -reticuline of the present invention comprises a protein consisting of a nucleotide sequence having 70% or more homology with respect to the nucleotide sequence of SEQ ID NO: 1 and having STORR enzymatic activity.
  • the gene 3 (hereinafter, also referred to as “the gene encoding STORR”), which is the encoding DNA, is composed of a nucleotide sequence having 70% or more homology with the nucleotide sequence of SEQ ID NO: 2 and is composed of CYP80Y2.
  • a protein consisting of gene 1 which is a DNA encoding a protein having enzymatic activity and a nucleotide sequence having 70% or more homology to the nucleotide sequence of SEQ ID NO: 3 and having enzymatic activity of oxidreductase is encoded.
  • It is characterized by including a step of expressing a protein having the above-mentioned oxidoreductase and a protein having the enzymatic activity of the oxidoreductase, and a step of producing (R) -reticulin from (S) -reticulin by the recombinant host cell. To do.
  • the method for producing (R) -reticulin of the present invention described above is a step of deleting the nucleotide sequence encoding the N-terminal hydrophobic region of the protein having the enzymatic activity of CYP80Y2 from the gene 1, the nucleotide sequence of SEQ ID NO: 4.
  • 5-aminolevulinate synthase 1 which is a DNA encoding a protein having a nucleotide sequence having 70% or more homology with the enzyme and having the enzymatic activity of 5-aminolevulinate synthase 1 (Also referred to as "encoding gene") is introduced into the host cell to express the protein having the enzymatic activity of 5-aminolevulinate synthase 1, and 70% or more of the nucleotide sequence of SEQ ID NO: 5 is used.
  • Gene 5 (hereinafter, also referred to as "gene encoding CPR"), which is a DNA encoding a protein having the same nucleotide sequence as CPR and having enzymatic activity of CPR, is introduced into the host cell. , At least one of the steps of expressing the protein having the enzymatic activity of CPR may be further included. As a result, (R) -reticuline can be efficiently produced even when a prokaryote is used as a host cell.
  • the method for producing (R) -reticulin of the present invention described above is a step of deleting the nucleotide sequence encoding the N-terminal hydrophobic region of the protein having the enzymatic activity of CYP80Y2 from the gene 1, SEQ ID NO: A gene 4 which is a DNA encoding a protein having a nucleotide sequence having 70% or more homology to the nucleotide sequence of 4 and having the enzymatic activity of 5-aminolevulinate synthase 1 is introduced into the host cell.
  • the step of expressing the protein having the enzymatic activity of 5-aminolevulinate synthase 1 and the enzyme of CPR which comprises a nucleotide sequence having 70% or more homology with the nucleotide sequence of SEQ ID NO: 5. It may further include the step of introducing the gene 5, which is the DNA encoding the protein having the activity, into the host cell to express the protein having the enzymatic activity of the CPR.
  • (R) -reticuline can be produced even more efficiently even when a prokaryote is used as a host cell.
  • the host cell may be a prokaryote.
  • the prokaryote may be Escherichia coli.
  • the amount of (S) -reticuline is finally below the detection limit, and the ratio of (R) -reticuline produced is apparently 100% of the total amount of reticuline.
  • the production method of the present invention can produce (R) -reticuline with extremely high conversion efficiency as described above even when a prokaryote is used as a host cell. Therefore, the production method of the present invention is practically useful because, for example, Escherichia coli, which is a prokaryote, can be applied as a host cell to produce (R) -reticuline.
  • the present invention it is possible to provide a method for producing (R) -reticuline from (S) -reticuline with extremely high conversion efficiency.
  • the amount of (S) -reticuline can be finally reduced to below the detection limit. Therefore, in the production method of the present invention, it is not necessary to carry out a process (optical resolution or the like) for obtaining only (R) -reticuline. Further, in the production method of the present invention, even if a prokaryote is used as a host cell, (R) -reticuline can be produced with high conversion efficiency as described above.
  • a thebaine production system applicable to Escherichia coli which is a practically useful prokaryote
  • a thebaine production system applicable to Escherichia coli which is a practically useful prokaryote
  • Opioid analgesics can be supplied.
  • the biosynthetic pathway of (R) -reticuline reconstructed in a host cell is shown.
  • It is the LC-MS analysis result which shows the production of (R) -reticuline in this embodiment.
  • homology means the degree of sequence similarity between two polypeptides or two polynucleotides, and the optimum state (sequence matching) over the region of the amino acid sequence or base sequence to be compared. Is determined by comparing the two sequences aligned to (maximum). The homology value (%) determines the number of sites in both aligned (amino acid or base) sequences that have the same amino acid or base, and then the number of sites is the amino acid in the sequence region to be compared. Alternatively, it is calculated by dividing by the total number of bases and multiplying the obtained numerical value by 100.
  • Algorithms for obtaining optimum alignment and homology include various algorithms usually available to those skilled in the art (eg, BLAST algorithm, FASTA algorithm).
  • Amino acid sequence homology is determined using, for example, sequence analysis software such as BLASTP, FASTA.
  • sequence analysis software such as BLASTP, FASTA.
  • the homology of the base sequence is determined by using software such as BLASTN and FASTA.
  • the "host cell” into which the gene is introduced in the method of the present invention is not particularly limited, and examples thereof include prokaryotes such as Escherichia coli and Bacillus subtilis, and eukaryotes such as yeast and filamentous fungi. Escherichia coli is preferable as the host cell in the present invention.
  • the gene When introducing a gene into a host cell, the gene may be introduced directly onto the host cell genomic DNA, but it is preferable to introduce a vector into which the gene is incorporated into the host cell. All the transgenes may be integrated into the same vector, or they may be integrated into two or more separate vectors.
  • the vector expresses the transgene integrated therein.
  • a plasmid that can autonomously replicate in the host cell or a vector constructed from a phage for gene recombination is suitable.
  • the vector preferably contains a replication initiation site suitable for the host cell to be introduced, a selectable marker, an expression control sequence such as a promoter, and a transcription termination signal (terminator sequence).
  • the plasmid vector include a pET vector system, a pQE vector system, a pCold vector system and the like when expressed in Escherichia coli, and a pYES2 vector system, a pYEX vector system and the like when expressed in yeast.
  • Selectable markers include antibiotic resistance genes such as ampicillin resistance gene, kanamycin resistance gene, and streptomycin resistance gene.
  • the expression control sequence is to control the expression of a gene consisting of the DNA sequence in a host cell when properly linked to the DNA sequence, that is, to induce and / or promote the transcription of the DNA sequence into RNA. , Or a sequence that can be suppressed.
  • the expression control sequence includes at least a promoter.
  • the promoter may be a constitutive promoter or an inducible promoter.
  • the expression vector used in the present invention can be prepared by adding an appropriate restriction enzyme recognition site to the end of a desired gene by a conventional method.
  • a conventionally known method can be used, and examples thereof include a calcium chloride method, an electroporation method, and a heat shock method.
  • the culture conditions of the recombinant host cell are not particularly limited as long as the recombinant cell grows well, all of the target group of proteins are expressed, and their respective functions or enzyme activities are exhibited. Specifically, the culture conditions may be appropriately selected in consideration of the nutritional and physiological properties of the host, and are usually carried out in liquid culture.
  • the carbon source of the medium used for culturing the recombinant host cell is not particularly limited as long as it is a substance that can be used by the host cell, and examples thereof include sugar and glycerol.
  • sugars include monosaccharides such as glucose, fructose and galactose, and disaccharides such as sucrose, lactose and maltose.
  • the nitrogen source include ammonium sulfate and casamino acid.
  • salts, specific amino acids, specific vitamins and the like can be used as desired.
  • Examples of the medium for culturing Escherichia coli include LB medium, 2 ⁇ YT medium, and M9 minimum medium.
  • Examples of the medium for culturing yeast include SC medium, SD medium, and YPD medium.
  • the culture temperature can be appropriately changed as long as the host cell grows, the target enzyme is expressed, and its activity is exhibited.
  • culture conditions of a temperature of 25 ° C. for 80 hours and a pH of 7.0 can be used.
  • culture conditions of a temperature of 30 ° C. for 60 hours and a pH of 5.8 can be used.
  • the produced (R) -reticuline can be confirmed by any means well known to those skilled in the art. Specifically, the reaction product and the target (R) -reticuline preparation can be identified by subjecting them to LC-MS and comparing the obtained spectra. It can also be confirmed by comparison by NMR analysis.
  • expressing means that the nucleic acid molecule constituting the gene is transcribed into at least an RNA molecule, and in the case of a gene encoding a polypeptide, it constitutes the gene. It means that a nucleic acid molecule is transcribed into an RNA molecule and the RNA molecule is translated into a polypeptide.
  • the expression level of the gene can be confirmed by a method known in the art, for example, Northern blot, quantitative PCR, or the like.
  • expressing means that transcription from a nucleic acid molecule encoding a polypeptide of the enzyme to an RNA molecule and translation from the RNA molecule to a polypeptide are normally performed. It means that an active enzyme is produced and exists inside or outside the cell.
  • the expression level of the enzyme can be confirmed by detection and quantification using a known method such as Western blotting or ELISA. It can also be confirmed by an assay for enzyme activity.
  • STORR is an enzyme derived from Papaver s Niniferum and is an epimerase that converts (S) -reticuline to (R) -reticuline.
  • STORR is a fusion polypeptide comprising a domain of CYP80Y2 and a domain of oxidoreductase.
  • SEQ ID NO: 9 is the amino acid sequence of STORR.
  • the "gene encoding STORR” used in the present invention is not limited to these, but may be, for example, the DNA described in any one of (a) to (c) below: (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 1; (B) Hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and has STORR enzymatic activity (eg, converting (S) -reticulin to (R) -reticulin).
  • A DNA consisting of the nucleotide sequence of SEQ ID NO: 1
  • B Hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and has STORR enzymatic activity (eg, converting (S) -reticulin to (R) -reticulin).
  • DNA encoding a protein that has epimerase activity to convert ;
  • C Consists of a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more with respect to the nucleotide sequence of SEQ ID NO: 1, and of STORR.
  • a DNA encoding a protein having enzymatic activity eg, epimerase activity that converts (S) -reticulin to (R) -reticrine).
  • the DNA consisting of the nucleotide sequence of (a) SEQ ID NO: 1 among the above (a) to (c) is preferably used.
  • CYP80Y2 is a P450 enzyme.
  • CYP80Y2 has, for example, the activity of oxidizing (S) -reticuline to produce 1,2-dehydroreticlinium.
  • the CYP80Y2 may be, for example, a domain of CYP80Y2 contained in the above-mentioned STORR separated from a domain of oxidoreductase.
  • CYP80Y2 obtained by adding starting methionine to the amino acid sequence shown in SEQ ID NO: 11 (Note that the amino acid sequence shown in SEQ ID NO: 11 is the one in which the N-terminal hydrophobic region is further deleted). Amino acid sequence of.
  • SEQ ID NO: 10 is the amino acid sequence of the N-terminal hydrophobic region deleted in the present embodiment.
  • the amino acid sequence of the N-terminal hydrophobic region was PTSSVVALLLALVSILSSVVV, but in this embodiment, it was deleted from the start codon.
  • the start codon sequence (atg) is inserted, and the stop codon sequence (taa) is introduced into the sequence immediately before the gene encoding oxidoreductase.
  • a gene encoding CYP80Y2 is obtained by adding a starting methionine to the amino acid sequence shown in SEQ ID NO: 11.
  • the "gene encoding CYP80Y2" used in the present invention is not limited to these, but may be, for example, the DNA described in any one of (a) to (c) below: (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 2; (B) Hybridizes with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and CYP80Y2 enzymatic activity (for example, by oxidizing (S) -reticrine, 1, 2 -DNA encoding a protein with (activity to produce dehydroreticrinium); (C) Consisting of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more homology with respect to the nucleotide sequence of SEQ ID NO: 2, and having CYP80Y2.
  • a DNA encoding a protein having enzymatic activity eg,
  • the DNA consisting of the nucleotide sequence of (a) SEQ ID NO: 2 among the above (a) to (c) is preferably used.
  • the "gene encoding CYP80Y2" used in the present invention may be one in which the nucleotide sequence encoding the N-terminal hydrophobic region of the protein having the enzymatic activity of CYP80Y2 is deleted.
  • the transmembrane region is deleted by deleting (cutting) the base sequence of the N-terminal hydrophobic region.
  • the deletion of the base sequence of the N-terminal hydrophobic region of the "gene encoding CYP80Y2" may be performed by deleting the base sequence of the N-terminal hydrophobic region of the "gene encoding STORR". That is, since the "gene encoding STORR" has a gene encoding CYP80Y2 on the N-terminal side and a gene encoding oxidoreductase on the C-terminal side, N of the "gene encoding STORR". If the nucleotide sequence of the terminal hydrophobic region is deleted, it means that the nucleotide sequence of the N-terminal hydrophobic region of the gene encoding CYP80Y2 has been deleted.
  • the "N-terminal hydrophobic region" of the "gene encoding CYP80Y2" refers to the “gene encoding CYP80Y2" (or “gene encoding STORR”). It is a hydrophobic region existing on the N-terminal side, and is, for example, the base sequence shown in SEQ ID NO: 8.
  • the oxidoreductase referred to in the present invention is an oxidoreductase having an enzymatic activity equivalent to that of the oxidoreductase domain contained in the above-mentioned STORR.
  • the oxid reductase may be, for example, a domain of the oxid reductase contained in the above-mentioned STORR separated from the domain of CYP80Y2.
  • SEQ ID NO: 12 is the amino acid sequence of the oxidoreductase thus obtained.
  • the "gene encoding oxidoreductase” used in the present invention is not limited to these, but may be, for example, the DNA described in any one of (a) to (c) below: (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 3; (B) Hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 and reduces the enzymatic activity of oxidoreductase (eg, 1,2-dehydroreticrinium).
  • A DNA consisting of the nucleotide sequence of SEQ ID NO: 3
  • B Hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 and reduces the enzymatic activity of oxidoreductase (eg, 1,2-dehydroreticrinium).
  • DNA encoding a protein having the enzymatic activity of eg, the activity of reducing 1,2-dehydroreticrinium to produce (R) -reticrine).
  • the DNA consisting of the nucleotide sequence of (a) SEQ ID NO: 3 among the above (a) to (c) is preferably used.
  • 5-Aminolevulinic acid synthase 1 is an enzyme that synthesizes 5-aminolevulinic acid using succinyl-CoA and glycine as substrates and pyridoxal phosphate as a coenzyme.
  • the "gene encoding 5-aminolevulinic acid synthase 1" used in the present invention is not limited to these, but is, for example, the DNA described in any one of (a) to (c) below.
  • DNA encoding a protein having (activity to synthesize 5-aminolevulinic acid) using pyridoxal phosphate as a coenzyme (C) Consists of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more homology with respect to the nucleotide sequence of SEQ ID NO: 4, and 5- DNA encoding a protein having the enzymatic activity of aminolevulinic acid synthase 1 (for example, the activity of synthesizing 5-aminolevulinic acid using succinyl-CoA and glycine as substrates and pyridoxal phosphate as a coenzyme).
  • a DNA consisting of the nucleotide sequence of (a) SEQ ID NO: 4 among the above (a) to (c) is preferably used.
  • a gene that is a DNA consisting of the nucleotide sequence of SEQ ID NO: 4 is called a HemA gene.
  • the Hema gene is a gene encoding 5-aminolevulinic acid synthase 1 of Rhodobacter sphaeroides.
  • CPR (NADPH-cytochrome P450 reductase 2) is an enzyme for transferring electrons from NADP to cytochrome P450 in microsomes. It also plays a role in transferring electrons to heme oxygenase and cytochrome B5.
  • the "gene encoding CPR" used in the present invention is not limited to these, but may be, for example, the DNA described in any one of (a) to (c) below: (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 5; (B) Hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 5 and transfers electrons from NADP to cytochrome P450 in the microsome.
  • DNA encoding a protein having enzymatic activity eg, activity of transferring electrons from NADP to cytochrome P450 in microsomes).
  • a DNA consisting of the nucleotide sequence of (a) SEQ ID NO: 5 among the above (a) to (c) is preferably used.
  • a gene that is a DNA consisting of the nucleotide sequence of SEQ ID NO: 5 is called an ATR2 gene.
  • the ATR2 gene is a gene encoding CPR of Arabidopsis thaliana.
  • the "stringent condition” refers to a condition in which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually about 6M urea, 0.4% SDS, 0.5 ⁇ SSC.
  • the DNA obtained by hybridization preferably has a high homology of 70% or more, more preferably 80% or more, and 90% or more homology with the DNA consisting of the nucleotide sequence of each SEQ ID NO:. It is more preferable to have 95% or more of homology.
  • the above gene can be obtained by PCR or hybridization technology well known to those skilled in the art. Further, the above gene may be artificially synthesized using a DNA synthesizer or the like. The sequence can be determined by a conventional method using a sequencer.
  • FIG. 1 shows the biosynthetic pathway of (R) -reticuline reconstituted in host cells in the present invention. Parentheses indicate the reaction in individual strains of each culture step.
  • (R) -reticuline is produced by converting (S) -reticuline to (R) -reticuline.
  • TYR tyrosinase
  • DDC dopadecarboxylase
  • MAO monoamine oxidase
  • 3,4-DHPAA 3,4-dihydroxyphenylacetaldehyde
  • SalS saltaridine synthase
  • SalR saltaridine reductase
  • Salutaridine acetyltransferase SPT (spontaneus).
  • FIG. 2 is a schematic view showing the STORR gene (FIG. 2 (a)) and the STORR gene (FIG. 2 (b)) in which the domain of CYP80Y2 and the domain of oxidoreductase are separated.
  • the method for producing (R) -reticuline of the present invention is to convert (S) -reticuline to (R) -reticuline, and (S) -reticuline is prepared in advance according to the conventional method.
  • the following steps 1 to 4 are carried out in this order.
  • step 1 described above can be omitted. That is, in the method for producing (R) -reticuline of the present invention, for example, the following steps 2 to 4 may be performed in this order.
  • a step (step 2) in which a gene encoding CYP80Y2 (gene 1) and a gene encoding oxidoreductase (gene 2) are inserted into a host cell to obtain a recombinant host cell.
  • the step of producing (R) -reticuline from (S) -reticuline by a recombinant host cell step 4).
  • the production method of (R) -reticuline when the host cell is a prokaryote (for example, Escherichia coli) is as follows.
  • the nucleotide sequence encoding the N-terminal hydrophobic region of the "gene encoding STORR” (gene 3) is deleted in advance (step A). More specifically, among the “genes encoding STORR", the N-terminal hydrophobic region of the gene encoding CYP80Y2 is deleted (cut), so that the transmembrane region is deleted and the start codon sequence ( Atg) has been inserted and the codon sequence (taa) has been introduced into the sequence immediately before the gene encoding the oxidoreductase.
  • the “gene encoding STORR” (gene 3) is divided into a “gene encoding CYP80Y2” (gene 1) and a “gene encoding oxidoreductase” (gene 2) (step B).
  • Gene encoding CYP80Y2 (gene 1), “gene encoding oxidoreductase” (gene 2), “gene encoding 5-aminolevulinate synthase 1” (gene 4), and “coding CPR”
  • a “gene” (gene 5) is introduced into a host cell to obtain a recombinant host cell (step C).
  • the introduction of these genes does not necessarily have to be done in this order. It may be done at the same time, or the order may be changed.
  • the protein having the enzymatic activity of CYP80Y2 encoded by the gene 1 the protein having the enzymatic activity of the oxidoreductase encoded by the gene 2, and the 5-aminolevulinate synthase 1 encoded by the gene 4
  • a protein having enzymatic activity and a protein having enzymatic activity of CPR encoded by gene 5 are expressed (step D).
  • the host cell produces (R) -reticuline from (S) -reticuline (step E).
  • step A the nucleotide sequence encoding the N-terminal hydrophobic region of the protein having the enzymatic activity of CYP80Y2 is deleted from the "gene encoding CYP80Y2" (gene 1) (step X). May be carried out.
  • the gene encoding CYP80Y2 shown in SEQ ID NO: 2 and the oxidative reductase shown in SEQ ID NO: 3 are encoded, which are separated from the gene encoding STORR shown in SEQ ID NO: 1 in the sequence listing (UniProtKB: P0DKI7).
  • the gene was used.
  • the HemA gene (UniProtKB: Q04512), which is a gene encoding 5-aminolevulinic acid synthase 1 shown in SEQ ID NO: 4, and the ATR2 gene (UniProtKB: Q9SUM3), which is a gene encoding CPR shown in SEQ ID NO: 5, ) was used.
  • Each gene has its codons optimized for E. coli.
  • Both genes were inserted into the NdeI-BamHI site of pET23a in order to realize regulation by the T7 promoter.
  • ligating genes first insert the gene to be connected to the NdeI-BamHI site of pET23a in front, and insert the gene to be connected to the back to the XhoI site located downstream of the NdeI-BamHI site, and then insert the expression vector. It was created.
  • a gene encoding oxidoreductase when inserted after a gene encoding CYP80Y2, an expression vector containing a gene encoding CYP80Y2 is inserted by first inserting the gene encoding CYP80Y2 into the NdeI-BamHI site of pET23a. CYP80Y2 / pET23a was created. Then, the gene encoding oxidoreductase was amplified by PCR using the primers of pr576 (SEQ ID NO: 6) and pr577 (SEQ ID NO: 7) shown in Table 1.
  • a gene encoding oxidoreductase is inserted by Infusion into the XhoI site located downstream of the NdeI-BamHI site of CYP80Y2 / pET23a, and an expression vector containing a gene encoding CYP80Y2 and a gene encoding oxidoreductase.
  • CYP80Y2-OxiRed / pET23a was created.
  • CYP80Y2Ncut-OxiRed / pET23a, ATR2 / pCDF23, Hema / pCDF23, and ATR2-Hema / pCDF23 were constructed as expression vectors by the same method as described above.
  • CYP80Y2Ncut-OxiRed / pET23a is an expression vector based on the pET23a plasmid containing a gene encoding CYP80Y2 in which the N-terminal hydrophobic region has been deleted and a gene encoding oxidoreductase.
  • Hema / pCDF23 is an expression vector based on the pCDF23 plasmid containing the Hema gene, which is a gene encoding 5-aminolevulinic acid synthase 1.
  • ATR2 / pCDF23 is an expression vector based on the pCDF23 plasmid containing the ATR2 gene, which is a gene encoding CPR.
  • ATR2-Hema / pCDF23 is an expression vector based on the pCDF23 plasmid, which contains the Hema gene, which is a gene encoding 5-aminolevulinic acid synthase 1, and the ATR2 gene, which is a gene encoding CPR.
  • the AN2534 strain is a strain in which CYP80Y2Ncut-OxiRed / pET23a is introduced into BL21DE3 strain, which is an Escherichia coli competent cell.
  • the AN4415 strain is a strain in which the empty vector pCDF23 is introduced into the AN2534 strain.
  • the AN4340 strain is a strain in which ATR2 / pCDF23 is introduced into the AN2534 strain.
  • the AN4341 strain is a strain in which HemA / pCDF23 is introduced into the AN2534 strain.
  • AN2051 is a strain in which ATR2-HemA / pCDF23 is introduced into the AN2534 strain.
  • Each E. coli strain is inoculated into LB medium (Difco) containing 50 mg / L ampicillin and 100 mg / L spectinomycin, cultured at 37 ° C. for 12 hours with shaking, and then TB containing 50 mg / L ampicillin and 100 mg / L spectinomycin.
  • Add 1/100 of the culture medium per 1 L: 12 g typtone (Difco), 24 g first extract (Diffco), 9.4 g K 2 HPO 4 , 2.2 g K 2 HPO 4 and 4 ml glycerol). It was. After culturing at 25 ° C.
  • FIG. 3 is an LC-MS analysis result showing the production of (R) -reticuline in the present embodiment.
  • FIG. 3A shows the result of introducing the gene encoding STORR shown in SEQ ID NO: 1 into the host cell with the full length (that is, without separating the gene encoding CYP80Y2 and the gene encoding oxidoreductase). is there.
  • FIG. 3B shows the result of introducing the gene encoding STORR shown in SEQ ID NO: 1 into a host cell by dividing it into a gene encoding CYP80Y2 and a gene encoding oxidoreductase.
  • FIG. 3 (c) is a result of stacking these samples, and is a diagram showing that the peak of FIG. 3 (a) and the peak of FIG. 3 (b) do not overlap.
  • (R) -reticuline could not be produced even if the gene encoding STORR was introduced into the host cell with the full length.
  • the gene encoding STORR is divided into a gene encoding CYP80Y2 and a gene encoding oxidoreductase and introduced into the host cell to obtain (R) -reticuline. It was shown that it can be obtained. That is, when the two domains of STORR were expressed separately, strong activity was obtained, and a sufficient amount (up to 100 ⁇ M) of (R) -reticuline could be produced.
  • a prokaryote was used as the host cell, but the present invention is not limited to this. That is, it is also possible to use eukaryotes as host cells. Since the P450 enzyme and the saltazine synthase (SalS) are functionally expressed in eukaryotes, it is possible to more easily produce (R) -reticuline from (S) -reticuline. For example, when a gene encoding CYP80Y2 and a gene encoding oxidoreductase are introduced into a eukaryote (yeast), it is considered that (R) -reticuline is produced from (S) -reticuline.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de production de (R)-réticuline consistant à obtenir une cellule hôte recombinée par insertion, dans une cellule hôte, d'un gène 1 étant composé d'une séquence nucléotidique ayant au moins 70 % d'homologie avec une séquence nucléotidique de SEQ ID NO : 2 et étant un ADN codant pour une protéine ayant une activité enzymatique de CYP80Y2, et un gène 2 étant composé d'une séquence nucléotidique ayant au moins 70 % d'homologie avec une séquence nucléotidique de SEQ ID NO : 3 et étant un ADN codant pour une protéine ayant une activité enzymatique d'oxydoréductase; à exprimer, dans la cellule hôte recombinée, la protéine ayant l'activité enzymatique de CYP80Y2 et la protéine ayant l'activité enzymatique de l'oxydoréductase ; et à produire de la (R)-réticuline à partir de la (S)-réticuline à l'aide de la cellule hôte recombinée.
PCT/JP2020/023561 2019-06-24 2020-06-16 Procédé de production de (r)-réticuline Ceased WO2020262107A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2021528257A JP7648038B2 (ja) 2019-06-24 2020-06-16 （ｒ）－レチクリンの生産方法
EP20832173.7A EP3988663A4 (fr) 2019-06-24 2020-06-16 Procédé de production de (r)-réticuline
US17/622,288 US12359233B2 (en) 2019-06-24 2020-06-16 Production method of (R)-reticuline

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019-116701 2019-06-24
JP2019116701 2019-06-24

Publications (1)

Publication Number Publication Date
WO2020262107A1 true WO2020262107A1 (fr) 2020-12-30

Family

ID=74060125

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/023561 Ceased WO2020262107A1 (fr) 2019-06-24 2020-06-16 Procédé de production de (r)-réticuline

Country Status (4)

Country Link
US (1) US12359233B2 (fr)
EP (1) EP3988663A4 (fr)
JP (1) JP7648038B2 (fr)
WO (1) WO2020262107A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116042550B (zh) * 2022-05-19 2025-07-25 湖南正清制药集团股份有限公司 清风藤碱合成酶及制备方法和在s-牛心果碱直接生成吗啡烷类生物碱的基本骨架中的用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012039438A1 (fr) 2010-09-22 2012-03-29 石川県公立大学法人 Procédé de production de benzylisoquinoléine-alcaloïde végétal
WO2015173590A1 (fr) * 2014-05-16 2015-11-19 The Unversity Of York Nouvelle protéine de fusion de cytochrome p450
WO2016207643A1 (fr) * 2015-06-24 2016-12-29 Sun Pharmaceutical Industries Australia Limited Production de noscapine
JP2017500024A (ja) 2013-12-04 2017-01-05 エピメロン インコーポレイテッドＥｐｉｍｅｒｏｎ Ｉｎｃ． （ｒ）−レチクリンおよびその前駆体を製造するための組成物および方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008067070A2 (fr) * 2006-10-19 2008-06-05 California Institute Of Technology Compositions et procédés pour produire des benzylisoquinoléines
US11124814B2 (en) * 2013-11-04 2021-09-21 The Board Of Trustees Of The Leland Stanford Junior University Benzylisoquinoline alkaloid (BIA) precursor producing microbes, and methods of making and using the same
US10463207B2 (en) 2016-05-20 2019-11-05 Linda Jane Bates One use portable toilet paper dispense system
WO2019028390A1 (fr) * 2017-08-03 2019-02-07 Antheia, Inc. Alcaloïde benzylisoquinoléine épimérases génétiquement modifiées et procédés de production des alcaloïdes benzylisoquinoléine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012039438A1 (fr) 2010-09-22 2012-03-29 石川県公立大学法人 Procédé de production de benzylisoquinoléine-alcaloïde végétal
JP2017500024A (ja) 2013-12-04 2017-01-05 エピメロン インコーポレイテッドＥｐｉｍｅｒｏｎ Ｉｎｃ． （ｒ）−レチクリンおよびその前駆体を製造するための組成物および方法
WO2015173590A1 (fr) * 2014-05-16 2015-11-19 The Unversity Of York Nouvelle protéine de fusion de cytochrome p450
WO2016207643A1 (fr) * 2015-06-24 2016-12-29 Sun Pharmaceutical Industries Australia Limited Production de noscapine

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AKIRA N ET AL.: "A bacterial platform for fermentative production of plant alkaloids", NAT COMMUN, vol. 2, 2011, pages 326, XP055165160, DOI: 10.1038/ncomms1327
AKIRA N ET AL.: "Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli", NAT COMMUN., vol. 7, 2016, pages 10390
NAKAGAWA A. ET AL.: "Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli", NATURE COMMUNICATIONS, 5 February 2016 (2016-02-05), XP055634246, DOI: 10.1038/ncomms10390 *
NAKAGAWA ET AL., NAT COMMUN, 2011
THILO W ET AL.: "Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein", SCIENCE, vol. 349, no. 6245, 2015, pages 309 - 312, XP055298478, DOI: 10.1126/science.aab1852
WINZER T. ET AL.: "Morphinan biosynthesis in opium poppy requires a P450- oxidoreductase fusion protein", SCIENCE, vol. 349, no. 6245, 17 July 2015 (2015-07-17), pages 309 - 312, XP055298478, DOI: 10.1126/science.aab1852 *

Also Published As

Publication number Publication date
EP3988663A4 (fr) 2023-08-02
JPWO2020262107A1 (fr) 2020-12-30
EP3988663A1 (fr) 2022-04-27
JP7648038B2 (ja) 2025-03-18
US20220251615A1 (en) 2022-08-11
US12359233B2 (en) 2025-07-15

Similar Documents

Publication Publication Date Title
Kim et al. 3-Amino-5-hydroxybenzoic acid synthase, the terminal enzyme in the formation of the precursor of mC7N units in rifamycin and related antibiotics
CN107690482B (zh) 用于2,4-二羟基丁酸的优化生产的经修饰的微生物
EP1292682A2 (fr) Procede de production de p-hydroxybenzoate en especes de pseudomonas et d'agrobacterium
WO2014048392A1 (fr) Méthode de production de composés stévioside au moyen de microorganismes
WO2011140344A1 (fr) Souches utilisées pour produire des flavonoïdes à partir du glucose
JPH10229885A (ja) 新規アルコールアルデヒド脱水素酵素
CA2876558C (fr) Procede de fabrication biologique d'acide cis-5-hydroxy-l-pipecolique
CN114075524B (zh) 阿魏酸生产工程菌、其建立方法及其应用
CN114717170B (zh) 异源合成黄酮类化合物的宿主细胞及其应用
CN114341350B (zh) 用于制备熊脱氧胆酸的方法
JP7648038B2 (ja) （ｒ）－レチクリンの生産方法
KR20130143507A (ko) 타이로신 고생산 균주에서의 인공대사 경로를 통한 4-쿠마린산, 카페인산 및 페룰린산의 생산 방법
JP2009142289A (ja) コバラミンを製造し得る生合成方法
WO2000058478A1 (fr) METHODE DE PRODUCTION MICROBIOLOGIQUE D'α-L-ASPARTYL-L-PHENYLALANINE
CN117903007B (zh) 一种亚精胺生物合成途径关键酶
EP3356514A1 (fr) Nouvelles souches bacteriennes pour la production de vanilline
CN112877349B (zh) 一种重组表达载体、包含其的基因工程菌及其应用
US7198932B1 (en) Gdp-4-keto-6-deoxy-d-mannose-3,5-epimerase-4-reductase gene derived from arabidopsis thaliana
EP1165799A1 (fr) Methode de production microbiologique d'alpha-l-aspartyl-l-phenylalanine
JP6635535B1 (ja) Ｅｆｐタンパク質を発現する大腸菌およびそれを用いたフラボノイド化合物製造方法
JP4759054B2 (ja) ニューロスポラ・クラッサ（Ｎｅｕｒｏｓｐｏｒａｃｒａｓｓａ）由来のＳ−アデノシルメチオニン−β−Ｎ−リジン−メチルトランスフェラーゼ、それをコードする遺伝子、同遺伝子を含むベクター及び宿主細胞、ならびに同宿主細胞を使用してトリメチルリジンを生産する方法
US20250320469A1 (en) Fusion protein and use for bioconverting molecules
CN111100848B (zh) 碳-碳环合酶及其编码基因与应用
KR20250080752A (ko) 수용성 메탄 모노옥시게나제를 이용한 최적의 탄화수소 산화물 생산방법
CN120310764A (zh) 一种融合蛋白及其在生产麦角硫因中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20832173

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021528257

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020832173

Country of ref document: EP

Effective date: 20220124

WWG Wipo information: grant in national office

Ref document number: 17622288

Country of ref document: US